1,4-naphthoquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-naphthoquinone

EC Number:

204-977-6

EC Name:

1,4-naphthoquinone

Cas Number:

130-15-4

Molecular formula:

C10H6O2

IUPAC Name:

1,4-naphthoquinone

Test material form:

solid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Preliminary test: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate.

Main test 1 and 2:
Without S9 mix: 0.153, 0.305, 0.610, 1.22, 2.44 and 4.88 µg/plate.
With S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate.

The main tests were conducted based on the results of the preliminary test using doses at which the maximum dose that microbial growth inhibition was clearly observed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Standard vehicle for tests of this type

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine hydrochloride monohydrate and 2-aminoanthracene.

Remarks:

Varying positive control substances depending on the strain/metabolic activation used.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation method (standard as per OECD guidance)

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: In the main test 3 plates/dose were used (triplicate). In the preliminary test 1 plate per dose was used (2 for controls).

MEASUREMENT OF COLONY NUMBER: Revertant colonies were measured using an automatic colony counter or manual counting.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn of the bacterial cells that have amino-acid requirements were observed by stereoscopic microscope and microbial inhibition (cytotoxicity) was judged by the relationship between the test substance treated plates and the solvent control plates.

Rationale for test conditions:

Pre-incubation method standard as per OECD guidance. Main experiment was repeated to confirm the positive result.

Evaluation criteria:

The substance was judged to be positive when it induced a dose dependant increase in the number of revertant colonies (mean) to a level equal to or greater than 2 fold of the solvent control value (mean) in any one of the tester strains with or without S9 mix, and when a dose dependant increase was reproducible. Other results were judged to be negative.

Statistics:

No statistical analysis was performed with the results in the study.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 78.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 10

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary test:
Cytotoxicity without S9 mix: 4.88 µg/plate or greater (all strains).
Cytotoxicity with S9 mix: 78.1 µg/plate or greater (all strains).

Main test 1:
Cytotoxicity without S9 mix: 2.44 µg/plate (all strains)
Cytotoxicity with S9 mix: 39.1 µg/plate or greater (TA100, TA1535, TA98, TA1537). 78.1 µg/plate (WP2urvA).
Precipitation: precipitation of the test substance was not observed in all tested strains both with and without S9 mix.

Main test 2:
Cytotoxicity without S9 mix: 2.44 µg/plate (all strains)
Cytotoxicity with S9 mix: 39.1 µg/plate or greater (TA100, TA1535, TA98, TA1537). 78.1 µg/plate (WP2urvA).
Precipitation: precipitation of the test substance was not observed in all tested strains both with and without S9 mix.

Sterility Test: Bacteria or fungus contamination was not observed in plates of the highest dose of the test substance solution, S9 mix or the 0.1 mol/L sodium phosphate buffer in the preliminary test and two main tests.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the substance was deemed to be potentially mutagenic as greater than a 2-fold number of revertant colonies was induced by the test substance compared with controls when considering the strain TA1537 with metabolic activation. This result was found in two experiments and the acceptability of the study was observed.